The present invention relates to new therapeutically active and selective inhibitors of the enzyme DPP-IV, pharmaceutical compositions comprising the compounds and the use of such compounds, and the manufacture of medicaments for treating diseases that are associated with proteins which are subject to inactivation by DPP-IV, such as type II diabetes and obesity.
Dipeptidyl peptidase-IV (DPP-IV), a serine protease belonging to the group of post-proline/alanine cleaving amino-dipeptidases, specifically removes the two N-terminal amino acids from proteins having proline or alanine in position 2.
Although the physiological role of DPP-IV has not been completely established, it is believed to play an important role in neuropeptide metabolism, T-cell activation, gastric ulceration, functional dyspepsia, obesity, appetite regulation, impaired fasting glucose (IFG) and diabetes.
DPP-IV has been implicated in the control of glucose metabolism because its substrates include the insulinotropic hormones glucagon like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP). GLP-1 and GIP are active only in their intact forms; removal of their two N-terminal amino acids inactivates them.
In vivo administration of synthetic inhibitors of DPP-IV prevents N-terminal degradation of GLP-1 and GIP, resulting in higher plasma concentrations of these hormones, increased insulin secretion and, therefore, improved glucose tolerance. Therefore, such inhibitors have been proposed for the treatment of patients with type II diabetes, a disease characterized by decreased glucose tolerance.
Unfortunately, the post-proline/alanine cleaving amino-dipeptidases are also implicated in the regulation of the immune system and inhibitors of these enzymes reportedly suppress immune responses. Thus, there is a risk that long-term treatment of type II diabetes with inhibitors of these enzymes may, as a side effect, lead to immuno-suppression.
However, with the recent discoveries of other post-proline/alanine cleaving amino-dipeptidases that share the same substrate and inhibitor specificity as DPP-IV, including DPP-IVb, Attractin, X and QPP, it has become clear that such inhibitors may inhibit multiple members of this group of enzymes. The precise physiological role of each of these post-proline/alanine cleaving enzymes is not well defined. Consequently, it is not clear what the physiological effect would be of inhibiting each of them separately, a subset, or all of them at the same time.
Diabetic dyslipidemia is characterized by multiple lipoprotein defects, including moderately high serum levels of cholesterol and triglycerides, small LDL particles, and low levels of HDL cholesterol. The results of recent clinical trials reveal beneficial effects of cholesterol lowering therapy in diabetic and nondiabetic patients, thus supporting increased emphasis on treatment of diabetic dyslipidemia. This need for intensive treatment of diabetic dyslipidemia was advocated by the National Cholesterol Education Program""s Adult Treatment Panel II.
Obesity is a well-known risk factor for the development of many very common diseases such as atherosclerosis, hypertension and diabetes. The incidence of obese people and thereby also these diseases is increasing throughout the entire industrialized world. Except for exercise, diet and food restriction no convincing pharmacological treatment for reducing body weight effectively and acceptably currently exist. However, due to its indirect but important effect as a risk factor in mortal and common diseases it will be important to find treatment for obesity or appetite regulation. Even mild obesity increases the risk for premature death, diabetes, hypertension, atherosclerosis, gallbladder disease and certain types of cancer. In the industrialized western world the prevalence of obesity has increased significantly in the past few decades. Because of the high prevalence of obesity and its health consequences, its prevention and treatment should be a high public health priority.
At present a variety of techniques are available to effect initial weight loss. Unfortunately, initial weight loss is not an optimal therapeutic goal. Rather, the problem is that most obese patients eventually regain their weight. An effective means to establish and/or sustain weight loss is the major challenge in the treatment of obesity today.
Thus there remains today a need in the art for compounds that are useful for inhibiting DPP-IV without suppressing the immune system.
Several compounds have been shown to inhibit DPP-IV, but all of these have limitations in relation to the potency, stability, selectivity, toxicity, and/or pharmacodynamic properties.
Such compounds have e.g. been disclosed in WO 98/19998, WO 00/34241, U.S. Pat. No. 6,124,305 (Novartis AG) and WO 99138501 (Trustees of Tufts University).
Thus, there is a need for novel DPP-IV inhibitors that are superior with respect to one or more of the above listed properties, and which will be useful for the treatment of conditions, which may be regulated or normalized by inhibition of DPP-IV.
The present invention provides novel 2-substituted unsaturated heterocyclic compounds, wherein a nitrogen atom in the heterocyclic ring is attached via an amide bond or a peptide bond to an amino acid or an amino acid derivative. These compounds are potent and selective inhibitors of DPP-IV, and are effective in treating conditions that may be regulated or normalized via inhibition of DPP-IV. The invention also concerns pharmaceutical compositions comprising the compounds, a method of inhibiting DPP-IV comprising administering to a patient in need of such treatment a therapeutically effective amount thereof, the compounds for use as a pharmaceutical, and their use in a process for the preparation of a medicament for treating a condition which may be regulated or normalized via inhibition of DPP-IV.
The term xe2x80x9cDPP-IVxe2x80x9d as used herein is intended to mean Dipeptidyl peptidase IV (EC 3.4.14.5; DPP-IV), also known as CD26. DPP-IV cleaves a dipeptide from the N terminus of a polypeptide chain containing a proline or alanine residue in the penultimate position.
The term xe2x80x9ctreatmentxe2x80x9d is defined as the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of a compound of the present invention to prevent the onset of the symptoms or complications, or alleviating the symptoms or complications, or eliminating the disease, condition, or disorder.
The term xe2x80x9cbeta cell degenerationxe2x80x9d is intended to mean loss of beta cell function, beta cell dysfunction, and death of beta cells, such as necrosis or apoptosis of beta cells.
The term xe2x80x9cC1-C10 alkylxe2x80x9d as used herein, alone or in combination, refers to a straight or branched, saturated hydrocarbon chain having from 1-10 carbon atoms such as but not limited to e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, sec. Butyl, isobutyl, tert. Butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 4-methylpentyl, neopentyl, 2,2-dimethylpropyl and the like.
The term xe2x80x9cC2-C10-alkenylxe2x80x9d used herein, alone or in combination, refers to a straight or branched, unsaturated hydrocarbon chain having from 2-10 carbon atoms and at least one double bond such as, but not limited to, vinyl, 1-propenyl, allyl, isopropenyl, n-butenyl, n-pentenyl and n-hexenyl and the like.
The term xe2x80x9cC2-C10-alkynylxe2x80x9d as used herein, alone or in combination, refers to an unsaturated hydrocarbon chain having from 2-10 carbon atoms and at least one triple bond such as but not limited to xe2x80x94Cxe2x89xa1CH, xe2x80x94Cxe2x89xa1CCH3, xe2x80x94CH2Cxe2x89xa1CH, xe2x80x94CH2xe2x80x94CH2xe2x80x94Cxe2x89xa1CH, xe2x80x94CH(CH3)Cxe2x89xa1CH and the like.
The term xe2x80x9cC1-10-alkoxyxe2x80x9d as used herein, alone or in combination is intended to include those C1-10-alkyl groups of the designated length in either a linear or branched or cyclic configuration linked through an ether oxygen having its free valence bond from the ether oxygen. Examples of linear alkoxy groups are methoxy, ethoxy, propoxy, butoxy, pentoxy and hexoxy. Examples of branched alkoxy are isoprpoxy, sec-butoxy, tert-butoxy, isopentoxy and isohexoxy. Examples of cyclic alkoxy are cyclopropyloxy, cyclobutyloxy, cyclopentyloxy and cyclohexyloxy.
The term xe2x80x9cC3-C10 cycloalkylxe2x80x9d as used herein refers to a radical of one or more saturated cyclic hydrocarbon having from 3-10 carbon atoms such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl and the like.
The term xe2x80x9cC3-C10 cycloalkanexe2x80x9d as used herein refers to a saturated cyclic hydrocarbon having from 3-10 carbon atoms such as, but not limited to, cyclopropane, cyclobutane, cyclopentane, cyclohexane, adamantane and the like.
The term xe2x80x9cC5-C10 cycloalkenylxe2x80x9d as used herein refers to a radical of one or more cyclic hydrocarbon having at least one double bond having from 5-10 carbon atoms such as, but not limited to, cyclopentenyl, cyclohexenyl and the like.
The term xe2x80x9carylxe2x80x9d as used herein includes carbocyclic aromatic ring systems. Aryl is also intended to include the partially hydrogenated derivatives of the carbocyclic systems.
The term xe2x80x9cheteroarylxe2x80x9d as used herein includes heterocyclic unsaturated ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sulphur such as furyl, thienyl, pyrrolyl. Heteroaryl is also intended to include the partially hydrogenated derivatives of the heterocyclic systems enumerated below.
The terms xe2x80x9carylxe2x80x9d and xe2x80x9cheteroarylxe2x80x9d as used herein refers to an aryl which can be optionally substituted or a heteroaryl which can be optionally substituted and includes phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N-hydroxytriazolyl, N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl, 3-anthracenyl), thiophenyl (2-thienyl, 3-thienyl), furyl (2-furyl, 3-furyl), indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3-triazol-1-yl, 1,2,3-triazol-2-yl 1,2,3-triazol-4-yl, 1,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2-thiazolyl, 4-thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl, 6-benzo[b]furanyl, 7-benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3-dihydro-benzo[b]furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo [b]furanyl), 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo[b]thiophenyl (2-benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6-benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo [b]thiophenyl (2-(2,3-dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo [b]thiophenyl), 4-(2,3-dihydro-benzo[b]thiophenyl), 5-(2,3-dihydro-benzo [b]thiophenyl), 6-(2,3-dihydro-benzo[b]thiophenyl), 7-(2,3-dihydro-benzo [b]thiophenyl), indolyl (1-indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl, benzimidazolyl (1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6-benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1-benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl, 7-benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b,f]azepine (5H-dibenz[b,f]azepin-1-yl, 5H-dibenz[b,f]azepine-2-yl, 5H-dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), 10,11-dihydro-5H-dibenz[b,f]azepine (10,11-dihydro-5H-dibenz[b,f]azepine-1-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-2-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-3-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-4-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-5-yl).
The present invention provides compounds of formula I 
wherein
at least one of the bonds in the five-membered ring is a double bond;
B is any alpha or beta amino acid connected to the ring with an amide or peptide bond;
or a salt thereof with a pharmaceutically acceptable acid or base.
In a preferred embodiment the invention provides compounds of formula II 
wherein
at least one of the bonds in the five-membered ring is a double bond;
R2 is H; C1-C10 alkyl optionally substituted with one or more R4 independently; C2-C10 alkenyl optionally substituted with one or more R4 independently; C2-C10-alkynyl optionally substituted with one or more R4 independently; C3-C10-cycloalkyl optionally substituted with one or more R4 independently; C5-C10 cycloalkenyl optionally substituted with one or more R4 independently; aryl optionally substituted with one or more R5 independently; or heteroaryl optionally substituted with one or more R5 independently;
R3 is H; C1-C10 alkyl optionally substituted with one or more R4 independently; C2-C10 alkenyl optionally substituted with one or more R4 independently; C2-C10-alkynyl optionally substituted with one or more R4 independently; C3-C10 cycloalkyl optionally substituted with one or more R4 independently; C5-C10 cycloalkenyl optionally substituted with one or more R4 independently; aryl optionally substituted with one or more R5 independently and/or fused to a C3-C10 cycloalkane; or heteroaryl optionally substituted with one or more R5 independently and/or fused to a C3-C10 cycloalkane;
R2 may be connected to R3 by a saturated or unsaturated bridge containing 1-3 carbon atoms, nitrogen atoms, oxygen atoms or sulphur atoms independently, or a valence bond, thus forming a ring, said ring may be fused to an aryl or heteroaryl, optionally substituted by one or more R5 independently;
R4 is cycloalkyl, aryl optionally substituted with one or more R5 independently; heteroaryl optionally substituted with one or more R5 independently; amino optionally substituted with one or more R6 independently; xe2x80x94SOxe2x80x94R6; xe2x80x94SO2xe2x80x94R6; xe2x80x94COxe2x80x94R6; xe2x80x94COOxe2x80x94R6, xe2x80x94CONHxe2x80x94R6; xe2x80x94CON(R6)2; xe2x80x94Oxe2x80x94R6; xe2x80x94Sxe2x80x94R6; carboxy; acetamido; cyano; nitro; halogen; hydroxy; trifluoromethyl; trifluoromethoxy; sulfamoyl; carbamoyl; hydroxymethyl;
R5 is halogen, C1-C10 alkyl, C1-C10 alkoxy, C1-C10 alkylamino, C1-C10 dialkylamino benzyl, benzyloxy, hydroxymethyl, nitro, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, N-hydroxyimino, cyano; carboxy; acetamido; hydroxy; sulfamoyl, carbamoyl;
R6 is C1-C10 alkyl, C2-C10 alkenyl, C2-C10-alkynyl, C3-C10-cycloalkyl, C5-C10 cycloalkenyl where any one of said alkyl, alkenyl, alkynyl, cycloalkyl, or cykloalkenyl may optionally be substituted with aryl optionally substituted with one or more R5 independently or heteroaryl optionally substituted with one or more R5 independently; benzyl, phenethyl; aryl optionally substituted with one or more R5 independently; or heteroaryl optionally substituted with one or more R5 independently
with the proviso that R2 and R3 cannot both be H;
or a salt thereof with a pharmaceutically acceptable acid or base.
In a preferred embodiment the invention provides compounds of formula III 
wherein
R2 is H; C1-C10 alkyl optionally substituted with one or more R4 independently; C2-C10 alkenyl optionally substituted with one or more R4 independently; C2-C10-alkynyl optionally substituted with one or more R4 independently; C3-C10-cycloalkyl optionally substituted with one or more R4 independently; C5-C10 cycloalkenyl optionally substituted with one or more R4 independently; aryl optionally substituted with one or more R5 independently; or heteroaryl optionally substituted with one or more R5 independently;
R3 is H; C1-C10 alkyl optionally substituted with one or more R4 independently; C2-C10 alkenyl optionally substituted with one or more R4 independently; C2-C10-alkynyl optionally substituted with one or more R4 independently; C3-C10 cycloalkyl optionally substituted with one or more R4 independently; C5-C10 cycloalkenyl optionally substituted with one or more R4 independently; aryl optionally substituted with one or more R5 independently and/or fused to a C3-C10 cycloalkane; or heteroaryl optionally substituted with one or more R5 independently and/or fused to a C3-C10 cycloalkane;
R2 may be connected to R3 by a saturated or unsaturated bridge containing 1-3 carbon atoms, nitrogen atoms, oxygen atoms, or sulphur atoms independently, or a valence bond, thus forming a ring, said ring may be fused to an aryl or heteroaryl, optionally substituted by one or more R5 independently;
R4 is cycloalkyl, aryl optionally substituted with one or more R5 independently; heteroaryl optionally substituted with one or more R5 independently; amino optionally substituted with one or more R6 independently; xe2x80x94SOxe2x80x94R6; xe2x80x94SO2xe2x80x94R6; xe2x80x94COxe2x80x94R6; xe2x80x94COOxe2x80x94R6, xe2x80x94CONHxe2x80x94R6; xe2x80x94CON(R6)2; xe2x80x94Oxe2x80x94R6; xe2x80x94Sxe2x80x94R6; carboxy; acetamido; cyano; nitro; halogen; hydroxy; trifluoromethyl; trifluoromethoxy; sulfamoyl; carbamoyl; hydroxymethyl;
R5 is halogen, C1-C10 alkyl, C1-C10 alkoxy, C1-C10 alkylamino, C1-C10 dialkylamino benzyl, benzyloxy, hydroxymethyl, nitro, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, N-hydroxyimino, cyano; carboxy; acetamido; hydroxy; sulfamoyl, carbamoyl;
R6 is C1-C10 alkyl, C2-C10 alkenyl, C2-C10-alkynyl, C3-C10-cycloalkyl, C5-C10 cycloalkenyl where any one of said alkyl, alkenyl, alkynyl, cycloalkyl, or cykloalkenyl may optionally be substituted with aryl optionally substituted with one or more R5 independently or heteroaryl optionally substituted with one or more R5 independently; benzyl, phenethyl; aryl optionally substituted with one or more R5 independently; or heteroaryl optionally substituted with one or more R5 independently
with the proviso that R2 and R3 cannot both be H;
or a salt thereof with a pharmaceutically acceptable acid or base.
In another preferred embodiment, the invention provides compounds of formula 
wherein
at least one of the bonds in the five-membered ring is a double bond;
R2 is H; C1-C10 alkyl optionally substituted with one or more R4 independently; C2-C10 alkenyl optionally substituted with one or more R4 independently; C2-C10-alkynyl optionally substituted with one or more R4 independently; C3-C10-cycloalkyl optionally substituted with one or more R4 independently; C5-C10 cycloalkenyl optionally substituted with one or more R4 independently; aryl optionally substituted with one or more R5 independently; or heteroaryl optionally substituted with one or more R5 independently;
R3 is H; C1-C10 alkyl optionally substituted with one or more R4 independently; C2-C10 alkenyl optionally substituted with one or more R4 independently; C2-C10-alkynyl optionally substituted with one or more R4 independently; C3-C10 cycloalkyl optionally substituted with one or more R4 independently; C5-C10 cycloalkenyl optionally substituted with one or more R4 independently; aryl optionally substituted with one or more R5 independently and/or fused to a C3-C10 cycloalkane; or heteroaryl optionally substituted with one or more R5 independently and/or fused to a C3-C10 cycloalkane;
R2 may be connected to R3 or R7 by a saturated or unsaturated bridge containing 1-3 carbon atoms, nitrogen atoms, oxygen atoms, sulphur atoms independently, or a valence bond, thus forming a ring, said ring may be fused to an aryl or heteroaryl, optionally substituted by one or more R5 independently;
R4 is cycloalkyl; aryl optionally substituted with one or more R5 independently; heteroaryl optionally substituted with one or more R5 independently; amino optionally substituted with one or more R6 independently; xe2x80x94SOxe2x80x94R6; xe2x80x94SO2xe2x80x94R6; xe2x80x94COxe2x80x94R6; xe2x80x94COOxe2x80x94R6, xe2x80x94CONHxe2x80x94R6; xe2x80x94CON(R6)2; xe2x80x94Oxe2x80x94R6; xe2x80x94Sxe2x80x94R6; carboxy; acetamido; cyano; nitro; halogen; hydroxy; trifluoromethyl; trifluoromethoxy; sulfamoyl; carbamoyl; hydroxymethyl;
R5 is halogen, C1-C10 alkyl, C1-C10 alkoxy, C1-C10 alkylamino, C1-C10 dialkylamino benzyl, benzyloxy, hydroxymethyl, nitro, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, N-hydroxyimino, cyano; carboxy; acetamido; hydroxy; sulfamoyl, carbamoyl;
R6 is C1-C10 alkyl, C2-C10 alkenyl, C2-C10-alkynyl, C3-C10-cycloalkyl, C5-C10 cycloalkenyl where any one of said alkyl, alkenyl, alkynyl, cycloalkyl, or cykloalkenyl may optionally be substituted with aryl optionally substituted with one or more R5 independently or heteroaryl optionally substituted with one or more R5 independently; benzyl, phenethyl; aryl optionally substituted with one or more R5 independently; or heteroaryl optionally substituted with one or more R5 independently;
R7 is H; C1-C10 alkyl optionally substituted with one or more R4 independently; C2-C10 alkenyl optionally substituted with one or more R4 independently; C2-C10-alkynyl optionally substituted with one or more R4 independently; C3-C10-cycloalkyl optionally substituted with one or more R4 independently; C5-C10-cycloalkenyl optionally substituted with one or more R4 independently; aryl optionally substituted with one or more R5 independently; heteroaryl optionally substituted with one or more R5 independently, halogen, C1-C10 alkoxy, C1-C10 alkylthio, C1-C10 alkylamino, C1-C10 dialkylamino, hydroxymethyl, nitro, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, N-hydroxyimino, cyano; carboxy; acetamido; hydroxy; sulfamoyl, carbamoyl;
with the proviso that the groups R2, R3, and R7 cannot all be H.
or a salt thereof with a pharmaceutically acceptable acid or base.
In another preferred embodiment, the invention provides compounds of formula V 
wherein
R2 is H; C1-C10 alkyl optionally substituted with one or more R4 independently; C2-C10 alkenyl optionally substituted with one or more R4 independently; C2-C10-alkynyl optionally substituted with one or more R4 independently; C3-C10-cycloalkyl optionally substituted with one or more R4 independently; C5-C10 cycloalkenyl optionally substituted with one or more R4 independently; aryl optionally substituted with one or more R5 independently; or heteroaryl optionally substituted with one or more R5 independently;
R3 is H; C1-C10 alkyl optionally substituted with one or more R4 independently; C2-C10 alkenyl optionally substituted with one or more R4 independently; C2-C10-alkynyl optionally substituted with one or more R4 independently; C3-C10 cycloalkyl optionally substituted with one or more R4 independently; C5-C10 cycloalkenyl optionally substituted with one or more R4 independently; aryl optionally substituted with one or more R5 independently and/or fused to a C3-C10 cycloalkane; or heteroaryl optionally substituted with one or more R5 independently and/or fused to a C3-C10 cycloalkane;
R2 may be connected to R3 or R7 by a saturated or unsaturated bridge containing 1-3 carbon atoms, nitrogen atoms, oxygen atoms, or sulphur atoms independently, or a valence bond, thus forming a ring, said ring may be fused to an aryl or heteroaryl, optionally substituted by one or more R5 independently;
R4 is cycloalkyl; aryl optionally substituted with one or more R5 independently; heteroaryl optionally substituted with one or more R5 independently; amino optionally substituted with one or more R6 independently; xe2x80x94SOxe2x80x94R6; xe2x80x94SO2xe2x80x94R6; xe2x80x94COxe2x80x94R6; xe2x80x94COOxe2x80x94R6, xe2x80x94CONHxe2x80x94R6; xe2x80x94CON(R6)2; xe2x80x94Oxe2x80x94R6; xe2x80x94Sxe2x80x94R6; carboxy; acetamido; cyano; nitro; halogen; hydroxy; trifluoromethyl; trifluoromethoxy; sulfamoyl; carbamoyl; hydroxymethyl;
R5 is halogen, C1-C10 alkyl, C1-C10 alkoxy, C1-C10 alkylamino, C1-C10 dialkylamino, benzyl, benzyloxy, hydroxymethyl, nitro, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, N-hydroxyimino, cyano; carboxy; acetamido; hydroxy; sulfamoyl, carbamoyl;
R6 is C1-C10 alkyl, C2-C10 alkenyl, C2-C10-alkynyl, C3-C10-cycloalkyl, C5-C10 cycloalkenyl where any one of said alkyl, alkenyl, alkynyl, cycloalkyl, or cykloalkenyl may optionally be substituted with aryl optionally substituted with one or more R5 independently or heteroaryl optionally substituted with one or more R5 independently; benzyl, phenethyl; aryl optionally substituted with one or more R5 independently; or heteroaryl optionally substituted with one or more R5 independently
R7 is H; C1-C10 alkyl optionally substituted with one or more R4 independently; C2-C10 alkenyl optionally substituted with one or more R4 independently; C2-C10-alkynyl optionally substituted with one or more R4 independently; C3-C10-cycloalkyl optionally substituted with one or more R4 independently; C5-C10 cycloalkenyl optionally substituted with one or more R4 independently; aryl optionally substituted with one or more R5 independently; heteroaryl optionally substituted with one or more R5 independently, halogen, C1-C10 alkoxy, C1-C10 alkylthio, C1-C10 alkylamino, C1-C10 dialkylamino, hydroxymethyl, nitro, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, N-hydroxyimino cyano; carboxy; acetamido; hydroxy; sulfamoyl, carbamoyl;
with the proviso that the groups R2, R3, and R7 cannot all be H.
or a salt thereof with a pharmaceutically acceptable acid or base.
A further preferred embodiment is represented by the compounds of the invention wherein:
R2 is H; C1-C10 alkyl optionally substituted with R4; C2-C10 alkenyl optionally substituted with R4; C2-C10-alkynyl optionally substituted with R4; aryl optionally substituted with one or more R5 independently; or heteroaryl optionally substituted with one or more R5 independently;
R2 may be connected to R3 or R7 by a saturated or unsaturated bridge containing 1-3 carbon atoms, nitrogen atoms, oxygen atoms, or sulphur atoms independently, or a valence bond, thus forming a ring, said ring may be fused to an aryl or heteroaryl, optionally substituted by one or more R5 independently;
Another preferred embodiment is represented by the compounds of the invention wherein R2 is H or C1-C10 alkyl optionally substituted with R4.
Another preferred embodiment is represented by the compounds of the invention wherein R2 is H.
Another preferred embodiment is represented by the compounds of the invention wherein:
R3 is H; C1-C10 alkyl optionally substituted with R4; C2-C10 alkenyl optionally substituted with R4; C2-C10-alkynyl optionally substituted with R4; C3-C10 cycloalkyl optionally substituted with R4; aryl optionally substituted with one or more R5 independently and/or fused to a C3-C10 cycloalkane; or heteroaryl optionally substituted with one or more R5 independently and/or fused to a C3-C10 cycloalkane;
Another preferred embodiment is represented by the compounds of the invention wherein:
R3 is H; C1-C10 alkyl optionally substituted with R4; or aryl optionally substituted with one or more R5 independently and/or fused to a C3-C10 cycloalkane.
Another preferred embodiment is represented by the compounds of the invention wherein R3 is C1-C10 alkyl optionally substituted with R4.
Another preferred embodiment is represented by the compounds of the invention wherein:
R4 is cycloalkyl; aryl optionally substituted with one or more R5 independently; heteroaryl optionally substituted with one or more R5 independently; xe2x80x94SOxe2x80x94R6; xe2x80x94SO2xe2x80x94R6; xe2x80x94COxe2x80x94R6; xe2x80x94COOxe2x80x94R6; xe2x80x94Oxe2x80x94R6; xe2x80x94Sxe2x80x94R6;
Another preferred embodiment is represented by the compounds of the invention wherein:
R4 is aryl optionally substituted with one or more R5 independently; heteroaryl optionally substituted with one or more R5 independently; xe2x80x94COxe2x80x94R6; xe2x80x94COOxe2x80x94R6; xe2x80x94Oxe2x80x94R6; xe2x80x94Sxe2x80x94R6;
Another preferred embodiment is represented by the compounds of the invention wherein R4 is aryl optionally substituted with one or more R5 independently;
Another preferred embodiment is represented by the compounds of the invention wherein R4 is xe2x80x94COOxe2x80x94R6, xe2x80x94Oxe2x80x94R6, or xe2x80x94Sxe2x80x94R6;
Another preferred embodiment is represented by the compounds of the invention wherein R5 is halogen, C1-C10 alkyl, C1-C10 alkoxy, C1-C10 alkylamino, C1-C10 dialkylamino, benzyl, or benzyloxy.
Another preferred embodiment is represented by the compounds of the invention wherein R5 is halogen, C1-C10 alkyl, or C1-C10 alkoxy.
Another preferred embodiment is represented by the compounds of the invention wherein R6 is C1-C10 alkyl, C2-C10 alkenyl optionally substituted with R4; C2-C10-alkynyl optionally substituted with R4; benzyl, aryl optionally substituted with one or more R5 independently, or heteroaryl optionally substituted with one or more R5 independently.
Another preferred embodiment is represented by the compounds of the invention wherein R6 is C1-C10 alkyl, benzyl, or aryl optionally substituted with one or more R5 independently.
The most preferred compounds of formula I wherein B represents an alpha-amino acid are the following:
(S,S) 1-(2-Amino-propionyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-butyryl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-3-methyl-butyryl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-3,3-dimethyl-butyryl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-4-methyl-pent-4-enoyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-3,3-diethyl-pentanoyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-2-cyclopentylacetyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-2-cyclohexylacetyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-2-cycloheptylacetyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-2-bicyclo[2.2.2]oct-1-yl-acetyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Adamantan-1-yl-2-amino-acetyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-2-phenylacetyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-2-(2,6 dimethylphenyl)acetyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-3,3-diphenyl-propionyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-(3(R)-methylpentanoyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-(4-methylpentanoyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2,6-Diamino-hexanoyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-6-dibenzylamino-hexanoyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-6-benzylamino-hexanoyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) [5-Amino-6-(2-cyano-2,5-dihydro-pyrrol-1-yl)-6-oxo-hexyl]-carbamic acid-tert-butyl ester
(S,S) [5-Amino-6-(2-cyano-2,5-dihydro-pyrrol-1-yl)-6-oxo-hexyl]-carbamic acid 9-H-fluoren-9-ylmethyl ester
(S,S) 4-Amino-5-(2-cyano-2,5-dihydro-pyrrol-1-yl)-5-oxo-pentanoic acid amide
(S,S) 4-Amino-5-(2-cyano-2,5-dihydro-pyrrol-1-yl)-5-oxo-pentanoic acid benzylamide
(S,S) 4-Amino-5-(2-cyano-2,5-dihydro-pyrrol-1-yl)-5-oxo-pentanoic acid benzyl ester
(S,S) 4-Amino-5-(2-cyano-2,5-dihydro-pyrrol-1-yl)-5-oxo-pentanoic acid-tert-butyl ester
(S,S) 1-(2-Amino-3-benzyloxy-propionyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-(4-methylsulfanyl-butyryl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(2-Amino-(3-phenylpropionyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-(Pyrrolidine-2-carbonyl)-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 6-{2-[2-(2-Cyano-2,5-dihydro-pyrrol-1-yl)-2-oxo-ethylamino]-ethylamino}-nicotino-nitrile
(S,S) 1-{2-[2-(5-Chloro-pyridin-2-ylamino)-ethylamino]-acetyl}-2,5-dihydro-1H-pyrrole-2 carbonitrile
(S,S) 1-{2-[2-(5-Trifluoromethyl-pyridin-2-ylamino)-ethylamino]-acetyl}-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-[2-(1-Hydroxymethyl-cyclopentylamino)-acetyl]-2,5-dihydro-1H-pyrrole-2-carbonitrile
(S,S) 1-{2-[2-(5-Nitro-pyridin-2-ylamino)-ethylamino]acetyl}-2,5-dihydro-1H-pyrrole-2carbonitrile, and
(S,S) 1-[2-(3-Isopropoxy-propylamino)-acetyl]-2,5-dihydro-1H-pyrrole-2-carbonitrile.
The most preferred compounds of formula I wherein B represents a beta-amino acid are the following:
1-(Piperidine-3-carbonyl)-2,5-dihydro-1-H-pyrrole-2-S-carbonitrile
1-(cis(2-Amino-cyclopenanecarbonyl))-2,5-dihydro-1-H-pyrrole-2-S-carbonitrile
1-(3-R-Amino-5-phenyl-pentanoyl)-2,5-dihydro-1-H-pyrrole-2-S-carbonitrile
1-(3-S-Amino-5-phenyl-pentanoyl)-2,5-dihydro-1-H-pyrrole-2-S-carbonitrile
1-(3-S-Amino-4-phenyl-butyryl)-2,5-dihydro-1-H-pyrrole-2-S-carbonitrile
1-(3-R-Amino-3-phenyl-propionyl)-2,5-dihydro-1-H-pyrrole-2-S-carbonitrile
1-(Morpholine-2-carbonyl)-2,5-dihydro-1-H-pyrrole-2-S-carbonitrile
1-(3-R-Amino-6-phenyl-hex-5-enoyl)-2,5-dihydro-1-H-pyrrole-2-S-carbonitrile
1-(3-R-Amino4-benzo[b]thiophen-2-yl-butyryl)-2,5-dihydro-1-H-pyrrole-2-S-carbonitrile
1-(3-R-amino-4-pyridin-3-yl-butyryl)-2,5-dihydro-1-H-pyrrole-2-S-carbonitrile
1-[3-S-Amino-4-(4-benzyloxy-phenyl)-butyryl]-2,5-dihydro-1-H-pyrrole-2-S-carbonitrile
1-[2-S-Pyrolidin-2-yl-acetyl)-2,5-dihydro-1-H-pyrrole-2-S-carbonitrile
1-[4-(2-Chloro-phenyl)-pyrrolidine-3-carbonyl]-2,5-dihydro-1-H-pyrrole-2-S-carbonitrile
1-(4-R-Phenyl-pyrrolidine-3-S-carbonyl)-2,5-dihydro-1-H-pyrrole-2-S-carbonitrile
or a salt thereof with a pharmaceutically acceptable acid or base.
The invention also relates to methods for preparing the above-mentioned compounds. These methods comprise (1) and (2) described below:
(1) Reacting an alpha amino acid or a beta-amino acid, suitably amino-protected with a standard protecting group such as Boc-, Fmoc-, CBz- or the like, with a 2-carbamoyl substituted unsaturated heterocyclic compound of formula VI, 
xe2x80x83under standard peptide coupling conditions to give an amide product; dehydrating the carbamoyl functionality of this material using standard dehydrating agents such as phosphorous oxycloride in pyridine or DMF, or trifluoroacetic acid anhydride, or the bromine/triphenylphospine adduct to give the nitrile, and to deprotect the amino group using standard chemical transformations to give the compounds of the invention.
(2) Reacting a 2-carbamoyl substituted unsaturated heterocyclic compound of formula VI, 
xe2x80x83with an alpha-halogenated carboxylic acid chloride, bromide, or anhydride to give compound of formula VII, 
xe2x80x83dehydrating the carbamoyl functionality of this material using standard dehydrating agents such as phosphorous oxycloride in pyridine or DMF, or trifluoroacetic acid anhydride, or the bromine/triphenylphospine adduct to give the nitrile, and reacting the nitrile compound with an appropriately substituted amine to give the compounds of the invention.
The compounds of the present invention may be prepared in the form of pharmaceutically acceptable salts, especially acid-addition salts, including salts of organic acids and mineral acids. Examples of such salts include salts of organic acids such as formic acid, fumaric acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid and the like. Suitable inorganic acid-addition salts include salts of hydrochloric, hydrobromic, sulphuric and phosphoric acids and the like. Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in Journal of Pharmaceutical Science, 66, 2 (1977) which are known to the skilled artisan.
Also intended as pharmaceutically acceptable acid addition salts are the hydrates which the present compounds are able to form.
The acid addition salts may be obtained as the direct products of compound synthesis. In the alternative, the free base may be dissolved in a suitable solvent containing the appropriate acid, and the salt isolated by evaporating the solvent or otherwise separating the salt and solvent.
The compounds of this invention may form solvates with standard low molecular weight solvents using methods known to the skilled artisan.
It is to be understood that the invention extends to all of the stereo isomeric forms of the claimed compounds, as well as the racemates.
A further aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for treating a condition that may be regulated or normalized via inhibition of DPP-IV.
Another aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for treatment of metabolic disorders.
Another aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for blood glucose lowering.
Another aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for treatment of type II diabetes
Another aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for the treatment of impaired glucose tolerance (IGT).
Another aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for the treatment of impaired fasting glucose (IFG).
Another aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for prevention of hyperglycemia.
Another aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for delaying the progression of impaired glucose tolerance (IGT) to type II diabetes.
Another aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for delaying the progression of non-insulin requiring type II diabetes to insulin requiring type II diabetes.
Another aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for increasing the number and/or the size of beta cells in a mammalian subject.
Another aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for treatment of beta cell degeneration, in particular apoptosis of beta cells.
Another aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for the treatment of disorders of food intake.
Another aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for the treatment of obesity.
Another aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for appetite regulation or induction of satiety.
Another aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for the treatment of dyslipidemia.
Another aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for treatment of functional dyspepsia, in particular irritable bowel syndrome.
A further aspect of the invention is a method for treating the conditions mentioned above by administering to a subject in need thereof an effective amount of a compound of the invention.
The invention furthermore relates to the use of a compound according to the present invention for the preparation of a medicament for use in the treatment of diabetes in a regimen that additionally comprises treatment with another antidiabetic agent.
In one embodiment of this invention, the antidiabetic agent is insulin or GLP-1 or any analogue or derivative thereof.
In another embodiment the antidiabetic agent is a non-peptidyl hypoglycemic agent, preferably an oral hypoglycemic agent.
Oral hypoglycemic agents are preferably selected from the group consisting of sulfonylureas, non-sulphonylurea insulin secretagogues, biguanides, thiazolidinediones, alpha glucosidase inhibitors, glucagon antagonists, GLP-1 agonists, potassium channel openers, insulin sensitizers, hepatic enzyme inhibitors, glucose uptake modulators, compounds modifying the lipid metabolism, compounds lowering food intake, and agents acting on the ATP-dependent potassium channel of the xcex2-cells.
Among the sulfonylureas, tolbutamide, glibenclamide, glipizide and gliclazide are preferred.
Among the non-sulphonylurea insulin secretagogues, repaglinide and nateglinide are preferred.
Among the biguanides, metformin is preferred.
Among the thiazolidinediones, troglitazone, rosiglitazone and ciglitazone are preferred.
Among the glucosidase inhibitors, acarbose is preferred.
Among the agents acting on the ATP-dependent potassium channel of the xcex2-cells the following are preferred: glibenclamide, glipizide, gliclazide, repaglinide.
In another aspect, the present invention includes within its scope pharmaceutical compositions comprising, as an active ingredient, at least one compound of the invention which inhibits the enzymatic activity of DPP-IV or a pharmaceutically acceptable salt or prodrug or hydrate thereof together with a pharmaceutically acceptable carrier or diluent.
Pharmaceutical compositions containing a compound of the invention of the present invention may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practise of Pharmacy, 19th Ed., 1995. The compositions may appear in conventional forms, for example capsules, tablets, aerosols, solutions, suspensions or topical applications.
Typical compositions include a compound of the invention which inhibits the enzymatic activity of DPP-IV or a pharmaceutically acceptable basic addition salt or prodrug or hydrate thereof, associated with a pharmaceutically acceptable excipient which may be a carrier or a diluent or be diluted by a carrier, or enclosed within a carrier which can be in the form of a capsule, sachet, paper or other container. In making the compositions, conventional techniques for the preparation of pharmaceutical compositions may be used. For example, the active compound will usually be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier which may be in the form of a ampoule, capsule, sachet, paper, or other container. When the carrier serves as a diluent, it may be solid, semi-solid, or liquid material that acts as a vehicle, excipient, or medium for the active compound. The active compound can be adsorbed on a granular solid container for example in a sachet. Some examples of suitable carriers are water, salt solutions, alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil, olive oil, gelatin, lactose, terra alba, sucrose, dextrin, magnesium carbonate, sugar, cyclodextrin, amylose, magnesium stearate, talc, gelatin, agar, pectin, acacia, stearic acid or lower alkyl ethers of cellulose, silicic acid, fatty acids, fatty acid amines, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, polyoxyethylene, hydroxymethylcellulose and polyvinylpyrrolidone. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax. The formulations may also include wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents. The formulations of the invention may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.
The pharmaceutical compositions can be sterilized and mixed, if desired, with auxiliary agents, emulsifiers, salt for influencing osmotic pressure, buffers and/or coloring substances and the like, which do not deleteriously react with the active compounds.
The route of administration may be any route, which effectively transports the active compound of the invention which inhibits the enzymatic activity of DPP-IV to the appropriate or desired site of action, such as oral, nasal, pulmonary, buccal, subdermal, intradermal, transdermal or parenteral, e.g., rectal, depot, subcutaneous, intravenous, intraurethral, intramuscular, intranasal, ophthalmic solution or an ointment, the oral route being preferred.
If a solid carrier is used for oral administration, the preparation may be tabletted, placed in a hard gelatin capsule in powder or pellet form or it can be in the form of a troche or lozenge. If a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.
For nasal administration, the preparation may contain a compound of the invention which inhibits the enzymatic activity of DPP-IV, dissolved or suspended in a liquid carrier, in particular an aqueous carrier, for aerosol application. The carrier may contain additives such as solubilizing agents, e.g., propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabenes.
For parenteral application, particularly suitable are injectable solutions or suspensions, preferably aqueous solutions with the active compound dissolved in polyhydroxylated castor oil.
Tablets, dragees, or capsules having talc and/or a carbohydrate carrier or binder or the like are particularly suitable for oral application. Preferable carriers for tablets, dragees, or capsules include lactose, corn starch, and/or potato starch. A syrup or elixir can be used in cases where a sweetened vehicle can be employed.
A typical tablet that may be prepared by conventional tabletting techniques may contain:
The compounds of the invention may be administered to a mammal, especially a human in need of such treatment, prevention, elimination, alleviation or amelioration of the various diseases as mentioned above, e.g., type II diabetes, IGT, IFG, obesity, appetite regulation or as a blood glucose lowering agent, and especially type II diabetes. Such mammals include also animals, both domestic animals, e.g. household pets, and non-domestic animals such as wildlife.
The compounds of the invention are effective over a wide dosage range. For example, in the treatment of adult humans, dosages from about 0.05 to about 1000 mg, preferably from about 0.1 to about 500 mg, per day may be used. A most preferable dosage is about 0.5 mg to about 250 mg per day. In choosing a regimen for patients it may frequently be necessary to begin with a higher dosage and when the condition is under control to reduce the dosage. The exact dosage will depend upon the mode of administration, on the therapy desired, form in which administered, the subject to be treated and the body weight of the subject to be treated, and the preference and experience of the physician or veterinarian in charge.
Generally, the compounds of the present invention are dispensed in unit dosage form comprising from about 0.05 to about 1000 mg of active ingredient together with a pharmaceutically acceptable carrier per unit dosage.
Usually, dosage forms suitable for oral, nasal, pulmonal or transdermal administration comprise from about 0.05 mg to about 1000 mg, preferably from about 0.5 mg to about 250 mg of the compounds admixed with a pharmaceutically acceptable carrier or diluent.
The invention also encompasses prodrugs of a compound of the invention which on administration undergo chemical conversion by metabolic processes before becoming active pharmacological substances. In general, such prodrugs will be functional derivatives of a compound of the invention which are readily convertible in vivo into a compound of the invention. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in xe2x80x9cDesign of Prodrugsxe2x80x9d, ed. H. Bundgaard, Elsevier, 1985.
The invention also encompasses active metabolites of a compound of the invention.
Chemical compounds are tested for their ability to inhibit the enzyme activity of purified CD26/DPP-IV. Briefly, the activity of CD26/DPP-IV is measured in vitro by its ability to cleave the synthetic substrate Gly-Pro-p-nitroanilide (Gly-Pro-pNA). Cleavage of Gly-Pro-pNA by DPP-IV liberates the product p-nitroanilide (pNA), whose rate of appearance is directly proportional to the enzyme activity. Inhibition of the enzyme activity by specific enzyme inhibitors slows down the generation of pNA. Stronger interaction between an inhibitor and the enzyme results in a slower rate of generation of pNA. Thus, the degree of inhibition of the rate of accumulation of pNA is a direct measure of the strength of enzyme inhibition. The accumulation of pNA is measured spectrophotometrically. The inhibition constant, Ki, for each compound is determined by incubating fixed amounts of enzyme with several different concentrations of inhibitor and substrate.
Materials:
The following reagents and cells are commercially available:
Porcine CD26/DPP-IV (Sigma D-7052), Gly-Pro-pNA (Sigma G0513).
Assay buffer: 50 mM Tris pH7.4, 150 mM NaCl, 0,1% Triton X-100.
Gly-Pro-pNA cleavage-assay for CD26:
The activity of purified CD26/DPP-IV is assayed in reactions containing:
70 ul assay buffer
10 ul inhibitor or buffer
10 ul substrate (Gly-Pro-pNA from a 0.1M stock solution in water) or buffer
10 ul enzyme or buffer
Reactions containing identical amounts of enzyme, but varying concentrations of inhibitor and substrate, or buffer as control, are set up in parallel in individual wells of a 96-well ELISA plate. The plate is incubated at 25xc2x0 C. and absorbance is read at 405 nm after 60 min incubation. The inhibitor constants are calculated by nonlinear regression hyperbolic fit and the result is expressed as inhibition constant (Ki) in nM.
The Zucker Diabetic Fatty (ZDF) rat model can be used to investigate the effects of the compounds of the invention on both the treatment and prevention of diabetes as rats of this sub-strain are initially pre-diabetic although develop severe type 2 diabetes characterized by increased HbA1c levels over a period of 6 weeks. The same strain can be used to predict the clinical efficacy of other anti-diabetic drug types. For example, the model predicts the potency and limited clinical efficacy of thiazolidinedione insulin sensitizer compounds.